Rapid, quantitative nonisotopic assay for telomerase activity in human tumors

¹ Clinical Biochemistry Unit, Department of Clinical Physiopathology, University of Florence, 50139 Florence, Italy.

² Clinical Laboratory Department, Azienda Ospedaliera Careggi, 50139 Florence, Italy.

³ Endocrinology Unit, Department of Clinical Physiopathology, University of Florence, 50139 Florence, Italy.

⁴ Institute of General Pathology, University of Florence, 50139 Florence, Italy.
^a Address correspondence to this author at: Clinical Biochemistry Unit, Department of Clinical Physiopathology, viale Pieraccini 6, 50139 Florence, Italy. Fax 39-55-4377290; e-mail c.orlando{at}dfc.unifi.it.

	Abstract

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Telomerase is a ribonucleoprotein enzyme that adds TTAGGG repeats onto human telomeres, preventing their shortening. The activation of this enzyme is an important step in cell immortalization and carcinogenesis and seems to represent a new and promising marker in cancer diagnosis and management. Telomerase activity is usually detected in cellular protein extract by the telomeric repeat amplification protocol (TRAP) assay, which can provide only a qualitative (presence/absence) evaluation. Here we present a modification of this method that can provide quantitative information without requiring time-consuming post-PCR procedures such as gel electrophoresis with radioactive materials and autoradiography. The detection and measurement of telomerase activity is performed by evaluating the amount of double-stranded DNA generated in the telomerase reaction and PCR amplification, with the use of the sensitive DNA fluorescent dye PicoGreen®. In a subset of tumors, the presence of telomerase activity was confirmed by the conventional TRAP assay. By this method we evaluated telomerase activity in unselected groups of breast (n = 15), ovarian (n = 12), endometrial (n = 12), gastric (n = 20), and renal (n = 12) carcinomas, in meningiomas (n = 8), and in pheochromocitomas (n = 10). The results indicate substantial differences of telomerase activity among cancer groups; however, a large variability among patients of the same group is observed. Kidney, ovarian, and breast carcinomas showed the highest mean values (31.8 ± 28.9, 29.2 ± 26.7, and 35.3 ± 15.9 ng DNA/µg protein, respectively, mean ± SD), whereas gastric and endometrial cancers had a lower activity (17.2 ± 8.8 and 13.5 ± 7.9 ng DNA/µg protein, respectively). Very low or no detectable telomerase activity was found in meningiomas (with the exception of one malignant atypical variant) and pheochromocitomas (9.7 ± 12.9 and 2.8 ± 2.1 ng DNA/µg protein, respectively). In conclusion, our method seems to be an accurate and reasonable procedure for measuring telomerase activity in human cancers.

	Introduction

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Immortality is one of the main characteristics of cancer cells. A large body of evidences revealed that the activation of telomerase, an enzyme that can elongate telomere ends, may contribute to the maintenance of cell immortality and to uncontrolled cell growth in cancer (1). The term telomeres defines the ends of chromosomes and their repeating sequences, highly conserved in most eukaryotes (2)(3). In humans the telomeric sequence TTAGGG is repeated thousands of times, reaching several kilobases in length (4)(5). Telomeres seem to stabilize chromosomes and prevent DNA degradation, as well as to provide a signal of cellular senescence (1)(6)(7). During each cycle of cell replication, telomeres are progressively shortened (8) because DNA polymerase complex does not replicate the very end of chromosomes (9)(10). Nondiseased somatic cells, when the telomere length is reduced to a critical point and cumulative loss impairs vital functions, exit from the cell cycle and become senescent (1).

Telomerase is a ribonucleoprotein complex that catalyzes the addition of telomeric repeats to the 3' end of chromosome DNA (4), thereby preventing the loss of telomeric sequences at each cell division. Because of its involvement in carcinogenesis, the activation of telomerase has been explored as a promising tool in cancer diagnosis and therapy (11)(12)(13)(14)(15).

An important improvement in telomerase detection was the development of the telomeric repeat amplification protocol (TRAP) assay (16). This assay is based on the PCR amplification of the in vitro telomerase reaction products. The addition to the PCR mixture of a radiolabeled nucleotide allows the revelation of telomerase activity on an autoradiographic film as a 6-bp ladder (16). This technique is highly sensitive and permits the revelation of telomerase activity even in limited amounts of cancer tissues or cultured cells. However, this approach is quite complex and cannot provide quantitative information on the effective activity of this enzyme. These limitations have not allowed clarification of the role of variable telomerase activity in determining the biological behavior of different human cancers.

Several changes of the conventional TRAP assay have been proposed to overcome this limitation, mainly based on the use of different primers to improve the specificity of PCR amplification (17), on the addition of an internal standard that permits the linearization of the TRAP assay (18), and on the use of fluorescent or biotinylated primers or probes to prevent the use of P and to obtain a semiquantitative evaluation of telomerase activity (19)(20)(21)(22). All of these techniques have provided interesting evolution of the assay; however, they included complex and time-consuming post-PCR procedures.

Here we present a modification of the TRAP assay based on the use of a sensitive fluorochrome (PicoGreen®) that selectively binds double-stranded DNA (23). Because the TRAP assay is based on a reaction that generates double-stranded DNA fragments starting from a protein extract and because the amount of generated DNA has to be considered proportional to the telomerase activity of the initial sample, we proposed that the estimated DNA concentration in post-PCR samples measured by PicoGreen can be considered quantitatively related to telomerase activity. Evidence is reported to demonstrate that this assay is rapid and simple and also provides sensitive, precise, and accurate measurements of telomerase activity in human tumor specimens.

	Materials and Methods

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samples collection and extraction
Telomerase activity was measured with the described method in unselected groups of breast (n = 15), ovarian (n = 12), endometrial (n = 12), and gastric (n = 20) carcinomas received in our laboratory for routine DNA index evaluation with cytometric analysis. In addition, the assay was also performed in some meningiomas (n = 8) and pheochromocytomas (n = 10). All surgical specimens were frozen in liquid nitrogen immediately after removal and stored at -80 °C until extraction. Telomerase activity was also measured in cell lines PC-3 and LNCaP from human prostate carcinoma. Telomerase was extracted from cancer samples and cell lines as described (16)(24)(25). Frozen tissue samples of ~100 mg were homogenized in 200 µL of 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate lysis buffer. For cell line extraction, pellets of cells were resuspended in 200 µL of 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. After 30 min of incubation on ice, the lysates were centrifuged at 16 000g for 20 min at 4 °C, and the supernatant was rapidly frozen and stored at -80 °C.

telomerase assay
Each sample was assayed for telomerase activity in duplicate, starting from protein extracts of the tissue or cell lines. A negative control, obtained after pretreatment of the sample with RNase, was also assayed for each specimen. The protein concentration was measured in each extract by the Bio-Rad Protein Assay (Bio-Rad Laboratories). An aliquot of extract containing 6 µg of protein was used for each duplicate. RNase (Boehringer Mannheim Italia) was used at 0.5 µg/assay for 30 min at 37 °C to inactivate telomerase. Each extract was assayed in 47.2 µL of reaction mixture containing 10 mmol/L Tris-HCl (pH 8.3), 50 mmol/L KCl, 4.5 mmol/L MgCl₂, 1 mmol/L each dNTP, 20 pmol of TAG-U primer (17), and 0.5 µmol/L T₄ gene 32 protein (Boehringer Mannheim Italia). After 60 min incubation at 30 °C for telomerase-mediated extension of TAG-U primer, the reaction mixture was heated at 90 °C for 3 min and then subjected to 60 PCR cycles of 95 °C for 30 s, 64 °C for 30 s, and 72 °C for 30 s, followed by 72 °C for 10 min after the addition of 2.8 µL of a second reaction mixture containing 20 pmol of CTA-R primer (17) and 0.3 µL of 5 U/µL of Taq Gold (Perkin-Elmer). Ten microliters of each PCR product was diluted with 490 µL of 10 mmol/L Tris-HCl, 1 mmol/L EDTA, pH 7.5, and then 500 µL of ultrasensitive fluorescent dye PicoGreen (Molecular Probes Inc.; 1:1000 diluted stock solution) was added. Fluorescence was measured in a spectrofluorophotometer RF-540 (Shimadzu) using standard wavelengths (excitation at 480 nm, emission at 520 nm). The DNA concentration was calculated for each sample on a calibration curve generated by dilutions of a control DNA (0–100 µg/L). The final DNA concentration of each sample was obtained by subtracting the DNA amount obtained in the same specimen after RNase treatment, as previously reported (17). Telomerase activity was calculated as the mean of duplicates and expressed in term of ng DNA/µg protein. In each assay we also evaluated a protein extract of a cell line (LNCaP) and a protein extract of a gastric tumor sample as positive controls. Human placental DNA was used as a negative control.

In a subset of cancer samples, the presence of telomerase activity was also tested by the conventional TRAP assay (16) with autoradiographic revelation of radiolabeled PCR products.

	Results

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kinetics of pcr reaction
To evaluate the influence of the number of PCR cycles on the amplification of telomerase-generated DNA fragments, samples with different telomerase activities were used to determine fluorescence at different number of PCR cycles. The quantity of amplified DNA was then calculated with PicoGreen in each sample. PCR generated parallel curves of product accumulation and a plateau effect after a variable number of cycles (Fig. 1 ).

View larger version (22K): [in this window] [in a new window]   Figure 1. Kinetics of PCR product accumulation of four samples with different telomerase activities (, , , and ) and a no-template control (). Telomerase activity was measured in each sample at five-cycle intervals (0–60 cycles). We obtained parallel kinetics with a typical plateau effect reached after a variable number of PCR cycles. The PCR profile of a sample with very high telomerase activity is reported in the inset. Each point is the mean of duplicate samples.

In the experiments we used a Taq polymerase (AmpliTaq Gold, Perkin-Elmer) that is particularly suitable for long amplification protocols and that reduces undesired primer dimer formation in the first cycles of PCR amplification, similar to a conventional hot start procedure (26). Because of the characteristics of this Taq polymerase, we chose to follow a protocol with 60 cycles of PCR amplification, according to the manufacturer's suggestions, to obtain the maximal sensitivity of the assay.

assay performance
The linearity of the assay was tested by measuring telomerase activity in different amounts of protein extract (1–6 µg) of a renal carcinoma with high telomerase activity, obtaining a good relationship with the quantities of DNA generated after PCR amplification (Fig. 2 ).

View larger version (20K): [in this window] [in a new window]   Figure 2. Linearity of telomerase assay. The measurement was performed starting from different protein amounts (1–6 µg) of a kidney carcinoma extract with high telomerase activity. A linear correspondence was obtained with the quantities of DNA obtained after PCR amplification, as measured by PicoGreen detection.

To test the precision of the proposed assay, we measured the telomerase activity in eight replicates of two samples with a different activity (26.1 and 50.4 ng DNA/µg total protein). The intraassay CV was 12.3% and 11.1%, respectively. The interassay precision in eight different assays was 14.5% and 15.3%, respectively.

The detection limit of the DNA calibration curve, measured by evaluating 2 SD over the mean of 10 replicates of the zero point, was 0.15 ng. Similarly we evaluated the detection threshold of PCR amplification by calculating 2 SD over the mean of 10 replicates of a no-template control containing all the components of PCR mixture but no DNA. This value was also calculated by measuring fluorescence in 10 replicates of a sample that had undergone the telomerase reaction followed by the addition of PCR reagents but without undergoing PCR cycling. These two experiments provided superimposable results; therefore, we could fix the real detection limit of the complete assay procedure at 4 ng DNA/µg protein. Samples with telomerase activity lower than this limit were considered as negative.

We tested the capacity of the proposed assay to discriminate between different-fold telomerase activity in reconstituted samples produced by serial dilutions of two specimens with different telomerase activity (27 and 4.4 ng DNA/µg protein, respectively). The results of this experiment are reported in Fig. 3 .

View larger version (22K): [in this window] [in a new window]   Figure 3. Accuracy of telomerase activity measurement. Reconstituted samples were prepared from two specimens with different telomerase activity (27 and 4.4 ng DNA/µg protein, respectively), according to the proportions indicated in the right panel, to have a constant amount of total protein (6 µg). Results were plotted against the expected values, and linear regression analysis was reported.

Furthermore, to test if our assay procedure generated a signal truly related to telomerase activity, in a subset of breast cancer samples, the reaction was also performed with the conventional TRAP assay based on P labeling (16). The resolution of corresponding PCR products by gel electrophoresis and autoradiography revealed the presence or the absence (after RNase treatment of the samples) of the telomerase activity in agreement to that found with the fluorescence method (Fig. 4 ).

View larger version (131K): [in this window] [in a new window]   Figure 4. Conventional TRAP assay of telomerase activity in five breast cancers with the autoradiographic revelation of 32P-labeled PCR-amplified products. Lanes 1, 3, 5, 7, and 10 are cancers; lanes 2, 4, 6, 8, and 9 are the corresponding RNase-treated samples.

telomerase activity in tumor samples and cancer cell lines
As shown in Fig. 5 , our method was able to measure variable telomerase activity in cancer samples we examined, whereas no activity was detectable in a few nondiseased control tissues of gastric mucosa (n = 5) and adrenal gland (n = 3; data not shown). On the other hand, telomerase activity was demonstrable in most renal (11 of 12, 92%), ovarian (9 of 12, 75%), breast (15 of 15, 100%), gastric (18 of 20, 90%), and endometrial carcinomas (10 of 12, 84%), even if the mean activities of the different groups are quite different (31.8 ± 28.9, 29.2 ± 26.7, 35.3 ± 15.9, 17.2 ± 8.8, and 13.5 ± 7.9 ng DNA/µg protein, respectively, mean ± SD). Meningiomas (4 of 8, 50%) and pheochromocitomas (1 of 10, 10%), tumors usually characterized by lower biological aggressiveness, showed low or not detectable telomerase activity (9.7 ± 12.9 and 2.8 ± 2.1 ng DNA/µg protein, respectively) with the exception of one malignant form of meningioma that showed very high telomerase activity. Furthermore, a large variability of telomerase activity was evident in renal, ovarian, and breast carcinomas, whereas gastric and endometrial cancers have an homogeneous distribution. Telomerase activity in prostate cancer cell lines PC-3 and LNCaP was 57 and 23 ng DNA/µg protein, respectively.

View larger version (24K): [in this window] [in a new window]   Figure 5. Telomerase activity in different cancer samples. The dotted line represents the lower detection limit of the assay.

	Discussion

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The development of the TRAP assay has allowed detection of the presence of telomerase activity in a wide variety of cancer specimens. These results seem to indicate that telomerase activation is the most widely expressed and specific cancer marker (14). Recent reviews reported a mean incidence of 85% of telomerase activation in malignant tumors (27); however, they also showed a wide variability of the percentage of telomerase positive cancers in the different neoplastic diseases (12). However, almost all of the reports that analyzed this aspect failed to give any information on the possible importance of variable telomerase activation and expression. The opportunity to have a quantitative evaluation of telomerase activity seems preliminary to a wider diffusion of this test for better classification of tumor malignancy and for its application as a prognostic and diagnostic marker.

Because the structure of the gene or genes involved in telomerase synthesis is only partially known (28)(29), data on the specific mRNA expression or immunological detection are still not available. Therefore, the measurement of the tissue expression of this enzyme must be done by evaluating its in vitro capability of de novo DNA synthesis, starting from a cellular protein extract. The use of the highly sensitive double-stranded DNA fluorescent dye PicoGreen therefore allows us to measure telomerase reaction and PCR amplification products, as previously proposed for studies on cell cycle modulation of telomerase activity in cultured cells (30). To eliminate nonspecific signals deriving from any combination of forward and reverse PCR primers, we used primers accurately designed to prevent primer dimer formation (17). Furthermore, the results for each sample were calculated in post-PCR products after the subtraction of fluorescence obtained in the corresponding specimen previously treated with RNase (17) to eliminate any possible nonspecific interference on the measurement of DNA specifically produced by telomerase reaction.

The proposed assay can be considered a practicable procedure to measure telomerase activity in human cancers or cell lines. A more accurate estimation of telomerase activity would require the presence, in the two-step assay procedure (primer extension and PCR amplification), of an internal standard to monitor unpredictable variability of the two reactions. However, as demonstrated by the evaluation of method performances, the precision, accuracy, and sensitivity are consistent with a practicable and high-throughput assay for the rapid comparison of telomerase activity in different cancers, without the requirement of complex and time-consuming techniques for post-PCR product analysis.

Using this procedure, we were able to detect telomerase activity in most malignant tumors examined in this study, with relevant differences among the different neoplasia. The mean telomerase activities found in kidney, ovary, and breast cancers were similar; however, single data points seem to indicate a wider distribution for the first two cancers. Interestingly, telomerase activity in breast cancer was found to be consistently higher, with no negative subjects. In the case of gastric and endometrium carcinomas, we observed lower telomerase activity, with a narrow distribution range. Cancers that in most cases are known to have a prevalent benign evolution, such as pheochromocitomas and benign meningiomas, showed very low or no detectable telomerase activity. The only case with high values was an atypical variant meningioma, as reported by others (12)(31).

At the moment we are not able to predict the clinical role of these findings, and we do not know if these results might have some relevance in predicting a different biological behavior among cancers with variable telomerase activation. In any case, our attempt is the first that tried to demonstrate that a large heterogeneity of telomerase activation does exist among cancer groups and patients. This effort is preliminary to a better knowledge on this specific topic and to the use of telomerase measurement in the clinical routine laboratory.

Some methodological problems still must be solved to improve the quality of the assay, in particular to obtain, through the introduction of an internal standard, an accurate control on the PCR amplification steps and an absolute quantitative assay of telomerase activity.

	Acknowledgments

 
This study was supported by a grant of the University of Florence and by financial support of Azienda Ospedaliera Careggi (Progetto Qualita). We also thank Zeneca s.p.a., Milan, Italy, for partial financial support of this research.

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